The voltage activated calcium channels (“VSCCs”) of vertebrates have been shown to be involved in a variety of different physiological processes including muscle contraction, insulin release from the pancreas, and neurotransmitter release in the nervous system (Greenberg D. Annals of Neurology, 1997;42:275-82; Catterall W. A., Trends in Neurosciences, 1993;16:500-506; Catterall W., Epstein P. N., Diabetologia, 35(Suppl 2:S23-33) 1992; Birnbaumer L., et al., Neuron., 1994:13; Rorsman P., et al., Diabete. Metab., 1994;20:138-145).
VSCCs are most highly expressed in excitable tissues including brain, skeletal muscle, and heart. They are multiprotein complexes composed of a central α1 pore-forming subunit variably associated with beta, gamma, and/or an α2δ subunit. Nine different functional classes of VSCCs have been described, based on biophysical and pharmacological studies. These functional classes are mainly determined by the α1 subunit composition. The beta, gamma, and α2δ subunits modulate channel function, affecting the kinetics of activation and inactivation, voltage-dependence, peak amplitude, and ligand binding. Walker N., De Waard M., Trends in Neurosciences, 1998;21(4):148-154.
A number of compounds useful in treating various diseases in animals, including humans, are thought to exert their beneficial effects by modulating functions of voltage-dependent calcium channels. Many of these compounds bind to calcium channels and alter cellular calcium flux in response to a depolarizing signal. However, a lack of understanding of the structure of channel subunits and the genes that code for them has hampered scientists both in discerning the pharmacology of compounds that interact with calcium channels and in the ability to rationally design compounds that will interact with calcium channels to have desired therapeutic effects. The lack of understanding is due in part to the fact that it has not been possible to obtain the large amounts of highly purified channel subunits that are required to understand, at the molecular level, the nature of the subunits and their interactions with one another, with the cell membranes across which the channels allow calcium ions to pass, with calcium and other ions, and with low molecular weight compounds that affect channel function.
Further, the lack of information on genes that code for calcium channel subunits has prevented the understanding of the molecular properties of the mature calcium channel subunits and their precursor proteins (i.e., the mature subunits with signal peptides appended to the amino-terminus) and the regulation of expression of calcium channel subunits. An understanding of these properties, and of how expression of calcium channel subunits genes is regulated, may provide the basis for designing therapeutic agents which have beneficial effects through affecting calcium channel function or concentration. Furthermore, the availability of sequences of genes coding for calcium channel subunits would make possible the diagnosis of defects, which might underlie a number of diseases, in genes coding for such subunits.
Expression experiments in Xenopus oocytes have demonstrated that in order to produce fully functional calcium channels, the α1 and α2δ subunits must both be expressed. Absence of the α2δ subunit results in a nonfunctional channel, even though the α1 subunit, through which ions flow, is fully expressed. Indeed, not only the ion flux through these channels, but the pharmacological properties of the α1 are different in the absence of the α2δ subunit. The α2δ subunit, therefore, is a critical component of VSCCs and one that must be studied if one is to better characterize VSCC function.
A detailed understanding of VSCC operation is beginning to reveal some mechanisms for interceding in the progression of diseases associated with abnormal VSCC functions. U.S. Pat. No. 5,618,720, which issued Apr. 8, 1997, references α1 and α2δ subunits and the polynucleotide sequences that encode the subunits. The publication, however, does not disclose any additional α2δ subunits and in light of the importance of the α2δ subunit, it can be understood that the identification and characterization of new α2δ subunits and the genes encoding these subunits would advance molecular genetic and pharmacological studies to understand the relations between the structure and the function of VSCCs.
Also, a further understanding of the biochemical mechanisms behind these subunits and their effect on mammals may lead to new opportunities for treating and diagnosing diseases related to abnormal (high or low) VSCC operation. Stated another way, a better understanding of the molecular mechanisms of VSCC operation will allow improved design of therapeutic drugs that treat diseases related to abnormal VSCC expression, and specifically abnormal α2δ expression.
The cDNAs, oligonucleotides, peptides, antibodies for the α2δ proteins, which are the subject of this invention, provide a plurality of tools for studying VSCC operations in various cells and tissues and for diagnosing and selecting inhibitors or drugs with the potential to intervene in various disorders or diseases in which altered α2δ expression is implicated. Such disease states affected include epilepsy and other seizure-related syndromes, migraine, ataxia and other vestibular defects (for review, Terwindt, G M et. Al., Eur J Hum Genet 1998 July-August; 6(4):297-307), chronic pain (Backonja M, JAMA 1998 Dec. 2;280(21):1831-6), mood, sleep interference (Rowbotham M, JAMA 1998 December 2;280(21):1837-42), anxiety (Singh et al., Psychopharmocology 1996 Sep. 127(1): 1-9), ALS (Mazzini L et. Al., J Neurol Sci 1998 October, 160 Suppl 1:S57-63), multiple sclerosis (Metz L, Semin Neurol 1998;18(3):389-95), mania (Erfurth A, et al., J Psychiatr Res 1998 September-October; 32(5):261-4), tremor (Evidente V G, et al., Mov Disord 1998 September; 13(5):829-31), parkinsonism (Olson W L, et al., Am J Med 1997 January; 102(1):60-6) substance abuse/addiction syndromes (Watson, W P et al., Neuropharmacology 1997 October; 36(10):1369-75), depression, and cancer, since at least one α2δ gene is located in a region of the genome which is thought to harbor an important tumor suppressor gene (Kok K., et al., Adv Cancer Res 1997;71:27-92).
The α2δ gene is also thought to play a part in proliferative diseases other than cancer, such as inflammation. Treatment with compounds which bind to α2δ lead to changes in the signal transduction mechanism of certain proteins. This includes altered levels of MEK (eg, MEK1 and MEK2) which activates the MAP kinase. Inhibitors of MEK appear to mimic the analgesic activities associated with the binding of gabapentin to α2δ. Activation of MAP kinase by mitogens appears to be essential for proliferation, and constitutive activation of this kinase is sufficient to induce cellular transformation.